Thelymphaticvascularsystemisessentialfortransportinginterstitialfluid,dietaryfat,andimmunecells.Defects inthesefunctionscontributetolymphedema,impairedlipidabsorption,obesity,abnormalimmunefunction,and cancermetastasis.Duringembryonicdevelopment,lymphangiogenesisisrobust,primarilydrivenbyvascular endothelialgrowthfactorC(VEGF-C)-mediatedactivationofVEGFR-3,amainVEGF-Creceptoronlymphatic endothelialcells(LECs).Emergingevidencehasshownthemetabolismofendothelialcellsiscriticalforvascular development.ChangesinECmetabolicpathwaysarefoundinpathologiessuchascanceranddiabetesaswell.But mostresearchhasbeenfocusedonbloodendothelialmetabolicpathways.Despiteafewrecentpioneeringstudies, knowledgeofLECmetabolismduringlymphangiogenesisislimited.Thereisanunmetneedtobridgetheknowledge gapbetweencellularmetabolismandlymphaticvasculardevelopment.Site-1protease(S1P),encodedby membrane-boundtranscriptionfactorpeptidase,site1(MBTPS1),isaserineproteaseintheGolgiapparatus.S1Pis akeyregulatorofcholesterolbiosynthesisbyproteolyticactivationofamembrane-boundlatenttranscriptionfactor, sterol-regulatoryelementbindingprotein2(SREBP2).Recently,wefoundthatmicewithinducibleendothelialcell- specificdeficiencyofS1P(iECMbtps1-/-,Mbtps1f/f;?Cdh5CreERT2)exhibitedseveresubcutaneouslymphedemaand defectivelymphaticvasculatureduringdevelopment.OurpilotexperimentsalsoshowedthatmicewithLEC-specific deficiencyofSREBP2(LECSrebf2-/-,Srebf2f/f;?Lyve1Cre)hadasimilarlymphaticvasculardefectduring development.ThesestronginvivopreliminarydatasupportthecentralhypothesisthatS1P/SREBP2-mediated cholesterolbiosynthesisisrequiredforlymphaticvasculardevelopment. WewilltestthecentralhypothesisthroughtwoAims:1)determinewhetherlymphaticendothelialS1P/SREBP2- mediatedcholesterolbiosynthesisisrequiredforlymphaticvasculardevelopment.WewillcharacterizeLECcellular defects,suchasdifferentiation,migration,andproliferation,ofS1PorSREBP2-deficientmiceatdifferentstagesof embryonicdevelopment.TheseinvivoanalyseswillbecomplementedbyinvitroassaysusingLECsisolatedfrom wild-type(WT)ormutantmiceaswellasprimaryhumanLECs;?2)determinemechanismsbywhichS1P/SREBP2- mediatedcholesterolbiosynthesisregulatelymphangiogenesis.Basedonourpreliminaryresults,wewillprimarily testthehypothesisS1P/SREBP2-mediatedcholesterolbiosynthesisisrequiredforsustainedVEGFR3signaling mainlybyinvitroassaysusingWTormutantLECsaswellashumanLECswithknockdownofS1P/SREBP2or functionalinhibitorstoS1PandSREBP2. Basedonstrongpreliminarydata,ourproposedstudywillrevealnovelinsightsintorolesofS1P-mediatedlipid metabolisminlymphaticvasculardevelopment.Ourstudymayleadtonoveltherapeuticopportunitiesfor pathologieswithlymphaticvasculardefects.